Neisseria gonorrhoeae is the etiologic agent for gonorrhea, one of the most prevalent infectious diseases in the U.S. Complications of gonorrhea include pelvic inflammatory disease, the leading cause of sterility in females. Despite the ability to effectively treat gonorrhea with antibiotics, the incidence of this disease remains high, suggesting that, in the absence of significant changes in sexual behavior, the most likely means of controlling gonorrhea will be by vaccination. This will require an understanding of the host immune response and of mechanism of pathogenesis of N. gonorrhoeae. One of the central themes of bacterial pathogenesis is that virulence factors are coordinately regulated and expression of those factors varies with environmental conditions. This project focuses on genes encoding outer membrane proteins (OMP) that are induced by anaerobiosis. The presence of antibody to the major anaerobically induced OMP, AniA, in sera from patients with gonorrhea indicates that this protein is expressed in vivo. The aniA gene has been sequenced, and there is evidence that its regulation may be complex; aniA may be part of a gonococcal virulence regulon. The Specific Aims are: 1) To determine the gene(s) and cis-acting elements involved in regulation of aniA; 2) To isolate other genes regulated by aniR, an activator of aniA expression; 3) To carry out structure/function analysis of the AniA protein. Biochemical and genetic experiments will be done to determine if the AniA protein is a respiration-linked nitrite reductase or is involved in regulation of nitrite reductase expression. FLAG peptide fusions to AniA will be used to define domains of the protein that are surface exposed, and deletion and site-specific mutagenesis will define essential regions of AniA. The role of AniA in invasion of HecIB cells will be determined.